CN107075554B - Method and device for detecting microorganisms in water samples - Google Patents

Abstract

The present invention relates to apparatus and methods for the detection of microorganisms present in, for example, seawater or other water samples. Means are provided for obtaining a water sample for testing, moving the water sample through a series of reservoirs, and binding the water sample to an indicator, such as a dye, that binds to the DNA of any microorganisms in the sample. Any binding of the indicator is measured by, for example, a spectrofluorometer or other suitable device for measuring the indicator.

Description

Method and device for detecting microorganisms in water samples

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to US Provisional Application No. 61/977,330, filed April 9, 2014.

technical field

The present invention relates to methods and apparatus for the continuous, automated, real-time detection of microorganisms in flowing water systems, such as those involving seawater, using biosensors and DNA staining techniques.

Background technique

In any system involving flowing water, the presence of microorganisms in the water can affect the system in a negative way. Examples of such problems include corrosion of instruments affected by microorganisms, blockage of systems or reservoirs, biofouling, etc. See US Pat. No. 8,525,130, incorporated herein by reference, which discusses problems caused by biofouling in desalination plants, and attempts to detect and analyze, for example, biofilms growing on plant equipment.

While there are many known methodologies for detecting the presence of microorganisms using biosensors, there is limited information available on the applicability of these methods to liquids such as natural water, reclaimed water, seawater, and the like. In this regard, US Patent 8,206,946 is also incorporated herein by reference.

US Patent 6,787,302, the disclosure of which is incorporated herein by reference, teaches the use of the commercially available dye "SYTO16" to detect viable cells in liquid samples. See also US Patent 8,206,946, Published US Application 20040191859 and PCT Application WO 1995 0191859, which disclose the use of fluorescent dyes to detect microorganisms. The contents of all these documents are incorporated herein by reference.

None of these references teach or disclose methods and/or apparatus for use in real-time analysis systems that can be used to automatically and continuously monitor water samples, such as seawater, for the presence of microorganisms .

The invention disclosed hereinafter relates to apparatus and methods that address the problems raised above.

SUMMARY OF THE INVENTION

The present invention relates to a method and apparatus for automatically monitoring microorganisms in a flowing water system (eg, seawater) on a continuous basis. Further, the present invention includes the incorporation of DNA staining technology into automated microbial sensors, thereby enabling detection of microorganisms by stained DNA, which is ubiquitous in microorganisms.

Description of drawings

Figure 1 shows an embodiment of the present invention.

Figure 2 shows the results of an experiment carried out in accordance with the present invention.

Figures 3a and 3b show further results.

Figure 4 shows still further results.

Detailed ways

Figure 1 shows an embodiment of the present invention. Referring to Figure 1, a conduit valve "101" connected to conduit 117 is depicted. This acts as a connector interface between the biosensor discussed below and the seawater injection sideline. A pressure regulator 102 is also described, which regulates the pressure of the water injected into the biosensor and ensures a steady flow into the sample chamber. As shown below, the water pressure should be below 10 bar.

The pipe arrangement with restrictor element "103" also facilitates the delivery of water in a steady, regular flow. Water flows through the plumbing arrangement into the sample chamber "104". The chamber has a constant overflow, which guarantees constant availability of fresh samples. Also shown is an auto-injector "105" calibrated to obtain an exact sample volume (eg 5ml), made easy by the two port injection valve "106" and dispensing valve "110". Fill valve 106 removes water from the sample chamber and indicator reservoir 107 . When the sample and DNA stain are mixed, the auto-injector "105" repeatedly draws the liquid, which is then transferred to the mixing chamber "108" to further ensure uniform mixing. More commonly, however, syringes are used to obtain liquid samples from, for example, the sample chamber, indicator reservoirs discussed below, reservoirs 111 and 112, and air samples from an air filter (not shown herein). The liquid or air can then be transferred to the sample chamber, indicator reservoir, mixing chamber discussed below, flow cell and water reservoir also discussed below. The positions of valve 110 and injection valve 106 control sample acquisition and distribution. Also shown is a flow cell "109" (which can be viewed as flowing through the cell), a first reservoir "111" containing, for example, distilled water, and a second reservoir "112" containing detergent, each reservoir The liquid container is provided with means "113" and "114" for drawing the respective raw materials into the working equipment also shown. In a preferred embodiment, the dispensing valve 110 is motorized. As shown in the embodiment of Figure 1, the dispensing valve has multiple ports (8 in this embodiment, each represented by a guide line) that control where the syringe draws fluid or air, and where to dispense the syringe contents when the syringe is emptied. Also shown herein is a portion of the housing arrangement 115 for protecting the device from the outside world. The sensor itself is not shown in this figure. After measurement, the waste of the sample is transferred by means 116 to, for example, a waste reservoir (not shown).

In operation, the device is automatically or manually turned to "on" mode. When this is done automatically, timers, computer controls, Internet or LAN connections, etc. can be used. When an automatic timer is used, it is set to turn on the computer using standard or custom software, or to program the computer that is part of the biosensor, or to be remotely controlled via an internal LAN. Automatic timer devices use the least amount of energy but other systems can also be used.

As can be seen from the above description, the apparatus of the present invention uses a dispensing system to transport and mix liquids (eg, water samples, dye solutions, detergents, and rinse water). In the described embodiment, a syringe is used, but the skilled person is aware of the possibility of other modes of dispensing and mixing.

Green I((N',N'-二甲基-N-[4-[(E)-(3-甲基-1,3-苯并噻唑-2-亚基)甲基]-1-苯基喹啉-1-um-2-yl]-N-丙基-1,3-二胺))与有着55％盐度的海水以1:10,000的比例混合。混合物以预定的时间进行培养，以使得指示剂渗透通过细菌的细胞膜并使指示剂与例如DNA进行结合。培养时长的选择可以变化，并且取决于所需的敏感度而增加。在下面的例子中，培养时间为40分钟。In operation, a sample of water, such as seawater, is removed from the sample chamber and mixed in a precise amount (as discussed below) with an indicator (eg, a DNA binding dye) in a predetermined but variable ratio. The ratio depends on many factors, including the nature of the indicator, the salinity of the liquid being tested, and other factors. In these instances,

Green I((N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-benzene quinoline-1-um-2-yl]-N-propyl-1,3-diamine)) was mixed with seawater having a salinity of 55% in a ratio of 1:10,000. The mixture is incubated for a predetermined period of time to allow the indicator to penetrate through the bacterial cell membrane and to bind the indicator to, for example, DNA. The choice of incubation time can vary and can be increased depending on the desired sensitivity. In the example below, the incubation time is 40 minutes.

After incubation, the sample is pumped to a flow cell that has been adapted to detect the indicator, eg, by fluorescence, eg, using an LED light source with an excitation wavelength of 490 nm. This parameter is used because of its short warm-up time and low energy requirements. The entry and emission of light can be controlled by filters to minimize interference, and the emitted light is measured by spectrometry at 520 nm. It is well known that LED performance and filters can be varied depending on the nature of the indicator used. Any data obtained by the system described herein can be stored in the sensor computer and can be obtained directly or remotely. Those skilled in the art will appreciate that the filters and LED wavelengths may vary depending on the dye used.

The length of operation and the number of measurements that can be taken in a given time period depends on a number of factors, such as the size of the reagent container.

Example 1

The following examples describe the application of the embodiments described above. A set run of 26 days was allowed, with 3 measurements a day prior to assay and reagent changes required.

The biosensor is placed on a flat surface and connected to a power supply (230V AC with live, neutral and ground). It should be noted that the system can be adapted to use solar panels, controls and batteries.

Further, the sensor is connected to the side wire and the pressure is less than 10 bar. The connection is facilitated by pushing at the connector, which facilitates the connection and replacement of transport devices that introduce water into the system.

In operation, current systems are designed to operate below 40°C. The included element will shut down the system if the temperature rises above 40°C. This feature is not required if the components used are not temperature sensitive, or if cooling means are introduced into the system.

The general manner in which the system operates is described in detail below; however, those skilled in the art will appreciate possible variations.

The biosensor first measures temperature and humidity to determine if there are limiting parameters (eg, temperature above 40°C, or humidity above 90%). As mentioned above, if this happens the device will shut down.

The sensor also provides information on whether any reagents need to be replenished.

If ambient conditions are suitable and the required amount of reagents are present, heat the spectrometer and flush the system with the sample. Next, a water sample and an amount of dye solution using a preset ratio are drawn into a syringe. These are mixed by repeatedly pumping the liquid into the mixing chamber (eg three times). After mixing, the sample is incubated, sent to the flow cell, and fluorescence detected.

Next, flush the system with detergent, distilled water, air, and then distilled water again. The system is then shut down and automatic instructions are entered as to when the process needs to be repeated.

The invention described herein reduces the time to analyze a water sample from weeks to hours.

Example 2

The water source was analyzed by the method proposed above. As can be seen from Figure 2, the ab initio microbial content was high; however, at the point indicated by the arrow mark (ten days after the start of the measurement), the biocide was added and the value was below the assay limit. (Note that the data in Figure 2 represent the correlation between fluorescent signal and manual counts).

Example 3

Long-term field trials of the invention described herein were conducted in Saudi Arabia. Figures 3a and 3b present this data, again in the form of a correlation between fluorescent staining and manual counts. A linear correlation allows it to be converted to cell number (cells per milliliter of seawater). Each system has a different conversion factor based on, inter alia, the chemical composition of the sample, dyes, and microbial size.

Example 4

In a long-term field trial, the microbial content of seawater was measured three times a day over a 4-month period. Figure 4 shows the results, providing useful information not only on the presence of microorganisms but also on periods of increased or decreased growth rates. For example, with biocide treatment, there were no detectable microorganisms, and the growth rate increased thereafter.

Other embodiments will be apparent to those skilled in the art and need not be repeated.

The terms and expressions used are used for description rather than limitation and their use is not intended to exclude any equivalents or parts of the features shown and described, it being understood that variations are may be within the scope of the present invention.

Claims (8)

1. Apparatus for detecting the presence of microorganisms in a water sample, comprising a biosensor connected by first valve means to means for providing a water sample to the biosensor, the biosensor further comprising pressure regulator means and restrictor means for regulating the flow of water into a sample chamber having first transport means for transporting a volume of sample to a mixing chamber; a DNA binding dye reservoir having a second means for transporting DNA binding dye to the mixing chamber, wherein the first and second means for transporting have been connected to an injection valve means for controlling the volume of sample and DNA binding dye; the mixing chamber having an injector device connected thereto, the injector device being operable to mix the sample and the DNA binding dye; means for receiving an analytical sample in fluid connection with the mixing chamber; and means for detecting said DNA binding dye in said means for containing said analytical sample.

2. The apparatus of claim 1, wherein the means for detecting the DNA binding dye is a spectrofluorimeter.

3. The apparatus of claim 1, further comprising a first reservoir means connected to the biosensor for delivering a quantity of a cleaning agent.

4. The apparatus of claim 3, further comprising a second reservoir means for delivering clean water to the biosensor.

5. The apparatus of claim 1, further comprising a waste disposal device connected to the mixing chamber.

6. A method for detecting the presence of a microorganism in a liquid sample, comprising transporting a liquid sample together with a DNA binding dye sample into the mixing chamber of the apparatus of claim 1 to form a mixture; incubating the mixture; and detecting the uptake of said DNA binding dye that has occurred as an indication of the presence of a microorganism.

7. The method of claim 6, wherein the liquid sample is seawater.

8. The method of claim 6, wherein the DNA binding dye is

green I。

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